Trypanosoma brucei gambiense group 2 experimental in vivo life cycle: from procyclic to bloodstream form

Trypanosoma brucei gambiense (Tbg) group 2 is a subgroup of trypanosomes able to infect humans and is found in West and Central Africa. Unlike other agents causing sleeping sickness, such as Tbg group 1 and Trypanosoma brucei rhodesiense, Tbg2 lacks the typical molecular markers associated with resistance to human serum. Only 36 strains of Tbg2 have been documented, and therefore, very limited research has been conducted despite their zoonotic nature. Some of these strains are only available in their procyclic form, which hinders human serum resistance assays and mechanistic studies. Furthermore, the understanding of Tbg2’s potential to infect tsetse flies and mammalian hosts is limited. In this study, 165 Glossina palpalis gambiensis flies were experimentally infected with procyclic Tbg2 parasites. It was found that 35 days post-infection, 43 flies out of the 80 still alive were found to be Tbg2 PCR-positive in the saliva. These flies were able to infect 3 out of the 4 mice used for blood-feeding. Dissection revealed that only six flies in fact carried mature infections in their midguts and salivary glands. Importantly, a single fly with a mature infection was sufficient to infect a mammalian host. This Tbg2 transmission success confirms that Tbg2 strains can establish in tsetse flies and infect mammalian hosts. This study describes an effective in vivo protocol for transforming Tbg2 from procyclic to bloodstream form, reproducing the complete Tbg2 cycle from G. p. gambiensis to mice. These findings provide valuable insights into Tbg2’s host infectivity, and will facilitate further research on mechanisms of human serum resistance.


Introduction
Trypanosoma brucei (Tb) is an extracellular protozoan parasite transmitted by an arthropod hematophagous vector: the tsetse fly (Glossina spp.) [16].Among the Tb species, the Tb brucei (Tbb) sub-species causes animal African trypanosomiasis or nagana in fauna.The human form of the disease, human African trypanosomiasis (HAT) or sleeping sickness is caused by the other two Tb sub-species: Tb rhodesiense (Tbr) and Tb gambiense (Tbg) [6].Tbr causes an acute form of the disease in East Africa, whereas Tbg develops into a chronic form in Central and West Africa.Tbg HAT was responsible of 87% of the reported cases in 2019-2020 and is targeted by the World Health Organization for interruption of transmission by 2030 [9].During the 1980s, the development of new analytical molecular methods allowed for the division of the Tbg subspecies into two groups.The most prevalent, genetically homogenous and monophyletic was group 1 (Tbg1) [11], invariably resistant to normal human serum (NHS) particularly thanks to the expression of the Tbg-specific glycoprotein (TgsGP) [2,29].In a recent review, group 2 (Tbg2) was defined as all human-infective Tb trypanosomes from West and Central Africa that do not fit into Tbg1 using various molecular markers [17].Tbg2, Tbb, and Tbr are obviously highly diverse lineages but Tbg2 is different from Tbr with a consistent lack of serum resistance associated gene (SRA) [12].If Tbg2 and its inconsistent resistance to lysis by human serum [21] does not appear to be a public health problem with only 36 strains referenced in the literature regarding the above definition, it represents a zoonotic form of HAT with a risk of transmission from animals to humans.In the current elimination context, it seems crucial to be able to detect such infections using adapted effective diagnosis and to determine if they are due to human serum resistance (HSR) trypanosomes or patient immunodeficiency (constitutive or transient) in order to implement adapted control strategies.Tbg2 stocks from different laboratories were gathered at UMR INTERTRYP (IRD/CIRAD, Montpellier, France) to study the HSR mechanisms and provide essential elements to anticipate the appearance of new mechanisms and to prevent possible phenomena of emergence [17].
Tb parasites have a multistage life cycle divided between the tsetse fly vector and a mammalian host.Along this life cycle, the parasite should continuously adapt to its surrounding environment.In the mammalian host, the bloodstream form (BSF) trypanosomes exist either in a proliferative long slender form or in a quiescent short stumpy form pre-adapted to the vector (Supplementary file 1).Following the infective blood meal, trypanosomes transform into their replicative procyclic form (PCF) in the tsetse fly midgut.Approximately one month after the infective meal, in a small proportion of tsetse flies (about 0.01% in natural conditions), trypanosomes colonize the salivary glands where they attach as epimastigote forms (EMF) [10].Trypanosomes finally differentiate into infectious metacyclic forms (MCF) that can be transmitted to the mammalian host during the next blood meal.
Most of the Tbg2 strains are only available in their PCF and cannot be tested for their resistance to NHS, and for the study of the mechanisms implied.Moreover, very little is known about the potential for infection of Tbg2 in tsetse and mammalian hosts.Some rare studies have been conducted using PCF of Tbg1 or Tbg2, but transmissions to a mammalian host were not successful or were not attempted [25,27].The objectives of the present experimental study were (i) to confirm that Tbg2 PCF can settle in tsetse flies and become infectious to mice, and (ii) to transform Tbg2 PCF to BSF for further studies on HSR.

Ethics for animal experiments
Mice were kept under strict ethical conditions according to the international guidelines for the care and use of laboratory animals.The experiments designed for this study were approved by the Regional Ethics Committee for Animal Experimentation CEEA-LR 36 under project number APAFiS #34149 and authorized by the French Ministry for Higher Education and Research.

Tsetse flies
In this study, tsetse flies were used from a colony of G. p. gambiensis originating from Burkina Faso and maintained at CIRAD (Montpellier, France).Only males were chosen, as they develop a higher proportion of salivary gland infections (mature infections) compared to females when experimentally infected with trypanosomes of the subgenus Trypanozoon [8,19,22].Due to several issues relative to fly physiology (natural death, fluctuating infecting meal feedings, low rate of trypanosome colonization in the midgut and of mature infection in the salivary glands) already described elsewhere [20,23], 165 tsetse flies were used assuming that this number would be sufficient to obtain mature infections after one month.Before the infection process, one wing was removed from each fly for security reason.

Experimental infection of tsetse flies
PCF trypanosomes (2.4 Â 10 8 ) were gently mixed with 40 mL of sheep blood heated to 31 °C.The infected blood was proposed to starved G. p. gambiensis teneral males through a silicone membrane [1].After the infective meal, tsetse flies were separated according to their blood-feeder status (blood meal visible in the abdomen or not).After 24 h, the process

Salivation assay and PCR
Thirty-five days after the infective meal (sufficient time to obtain mature infection in the salivary glands [25,30]), surviving tsetse flies were individually subjected to a salivation test.Each fly was allowed to salivate into a drop of phosphatebuffered saline-glucose (PSG) on warmed slides (37 °C) [4,14] and immediately placed into an individual cage.The salivate was recovered from the slide in 25 lL of sterile water (Figure 1).To identify flies whose saliva carried trypanosomes, DNA was extracted from each collected saliva and analyzed by TBR1/2 PCR, as already described [25].Flies with PCR-positive saliva were then grouped in different cages according to PCR signal intensity (strong, medium, or weak) (Figure 1).Flies with PCR-negative saliva and flies whose salivate could not be recovered because flies refused to salivate were euthanized and dissected for microscopic observation (Â400).

Monitoring and dissection of the tsetse flies
Midguts of all flies found dead during the process were dissected from day 5 (time needed to observe parasite colonization of the midgut) to day 19 post-infection (pi).From day 20 pi, the salivary glands were also dissected (assuming that no trypanosomes can be found in the salivary glands before this time).All the dissected midguts and salivary glands were examined for trypanosomes by phase contrast microscopy at 400Â magnification.

Infection of mice
At day 39 and 42 pi, each group of flies with PCR-positive saliva was fed twice (3 days apart) on the belly of anesthetized female BALB/c mice previously immuno-suppressed with 0.15 mL of cyclophosphamide (ENDOXAN, 20 mg/mL) injected subcutaneously (Figure 1).A different mouse was assigned to each group of flies.The objective of this sorting was to maximize the success of infection of one of the mice.Tsetse were starved for three days prior to the mice blood meal to increase sting probability.The parasitemia of the mice was then determined daily by microscopy using the rapid "matching" method [15] on a drop of blood collected from the tail of each mouse (Figure 1).
The mice-fed surviving flies were euthanized and dissected for microscopic (Â400) observation of the midgut and salivary glands.

Results and discussion
Molecular screening of tsetse flies with trypanosomes in their salivary glands Thirty-five days after tsetse fly infection, 80 flies out of 165 were still alive (Figure 2).Out of them, 78 were tested for the detection of trypanosomes in saliva by PCR and 43 (55%) showed PCR-positive saliva.Out of these 43 flies, 9, 12, and 22 exhibited a strong, medium, and weak PCR signal, respectively (Supplementary file 2).Between the beginning of the collection of the saliva and the results of the PCR analysis, 8 flies died.The remaining 35 flies were grouped into four cages: two containing 9 flies each with weak PCR signal, one containing 11 flies with medium PCR signal, and one containing 6 flies with strong PCR signal (Figure 2).All the PCR-negative flies were euthanized and dissected for microscopic observation of the midgut and salivary glands.

Monitoring of tsetse fly infection
Throughout this experiment, 115 flies could be dissected of which 11 (9.6%) showed parasites in their midgut only, and six (5.2%) in both their midgut and salivary glands.Among the flies showing trypanosomes in their salivary glands that were fed on mice, none were found in cage No. 1, one was from cage No. 2, one from cage No. 3 and two from cage No. 4, in line with the molecular analysis.
The number of mature infections identified by the PCR analysis of the flies' saliva (n = 43) was much higher than that determined by microscopic observation of the salivary glands (n = 6).These data are in the range found with previous observations from other studies (with maximum 10% flies found positive in SG) [25][26][27].Because of its high sensitivity, PCR from saliva may offer a better view of mature infections as it is sometimes challenging to observe trypanosomes in salivary glands.However, for most of the PCR-positive flies for trypanosomes in their saliva, no trypanosomes were  Additionally, because the recovery of salivary glands is not easy during fly dissection, salivary glands may not always be available for observation.This was the case for three of the flies dissected at day 36 pi: trypanosomes were observed in the midgut only, but the salivary glands could not be recovered for technical reasons.Therefore, the percentage of flies with mature infection may have been underestimated through microscopy techniques.

Monitoring infection in mice
Once the trypanosome PCR-positive flies from the four cages had been fed on mice, parasitemia was monitored daily.Three mice out of the four developed an infection two to four days after the blood meal (Figure 2 and Supplementary file 3).The resulting HTAG107-1 BSF trypanosomes were collected from the infected mice by cardiac puncture and supplemented with 50% glycerol before being stored in liquid nitrogen.Dissection and observation of the salivary glands of the flies used to bite the mice showed that two flies were positive for trypanosomes in cage No. 4, one in cage No. 3, and one in cage No. 2. No salivary gland-positive flies were detected in cage No. 1, which is congruent with the absence of infection in mouse 1.

Advantages and drawbacks to reconstitute a PCF to BSF experimental in vivo life cycle
From 165 flies fed with a single meal of sheep blood mixed with cultivated PCF trypanosomes, at least 6 flies with mature infections after one month were obtained.
We succeeded in infecting mice from infected tsetse flies.This achievement is partly due to the large size of the starting sample (n = 165) and to the starvation of the flies before the infective meal.Post-dissection of the flies used to infect the mice showed that a mouse only needs to be bitten by one fly with mature infection to become infected.This success also confirms that Tbg2 group strains can settle in the tsetse fly and infect a mammalian host.
For strains only available as PCF, the results of this experiment made it possible to obtain the bloodstream form that can be evaluated for resistance to NHS.However, this experiment is time consuming, requires great technical effort and is not in line with current animal ethics principles (3R rulereplace, reduce, refine).For this reason, if the passage from PCF to BSF is the only result desired, in vitro plasmid methods should be preferred [24].
While HAT elimination seems a realistic goal for 2030, we advocate for improving knowledge of the Tbg strains that are still circulating, even at a low, almost undetectable level.In several HAT foci, parasitemia observed in human or in animal reservoirs is very low [7] and hinders deep genotypical and phenotypical characterization.Isolating strains and mastering a transmission cycle makes it possible to collect data that are not available otherwise, for instance to account for differences in pathogenicity and virulence to humans over natural cycles [5].This is particularly interesting in the case of Tbg2 strains.They have been found to be resistant or partially resistant to the NHS, but their ability to maintain NHS resistance capacity after cycling in animals is unknown.Deciphering the nature of the resistance to NHSconstitutive (as is the case for Tbg1) or conditional (which can be lost after several vector/animal cycles)is keystone information for the effective and sustainable elimination of sleeping sickness.Indeed, elimination of HAT will lead to a decrease in acquired immunity in populations, which could create major concerns for more susceptible populations if they are again exposed to strains with constitutive resistance [17].
Finally, it remains difficult to reproduce complete cyclical transmission (from infection of the tsetse fly to transmission to the host by the tsetse fly) of Tbg2 and even Tbg1 because of the low rate of mature infections of Tbg [20].Two other studies succeeded using clones of Tbg2 BSF and Glossina morsitans [13,18].In this study, we provide evidence of an effective in vivo protocol to transform Tbg2 PCF to BSF by experimentally reproducing the complete Tbg2 cycle from G. p. gambiensis to the mouse.

2 P.
Juban et al.: Parasite 2024, 31, 15 of infective meal was repeated with the non-gorged tsetse flies to ensure maximum infection rate success.Flies were then fed with uninfected sheep blood, three days a week for 35 days.

Figure 2 .
Figure 2. Diagram summarizing the main results of the experimental infection of flies and mice with Trypanosoma brucei gambiense group 2 HTAG-107 strain.(*x: number of flies found positive in salivary glands by microscopic observation after dissection).

4 P.
Juban et al.: Parasite 2024, 31, 15 observed in the dissected salivary glands, despite diligent microscopic observation.Part of the PCR-positive saliva could result from flies only infected in the midgut, whose saliva may also contain regurgitated gut contents including trypanosome DNA.